Liquid crystalline polymers (LCPs) are a class of thermoplastics that exhibit a unique combination of properties including good flow, high temperature performance, low mold shrinkage, and excellent chemical resistance which allow them to be useful in many markets. 4-hydroxybenzoic acid (para-hydroxybenzoic acid, PHB) is a key monomer used in the production of LCPs. The cost of this monomer (approximately $2.40/lb) is a major portion of the high cost of LPCS, and limits the commercial applications for which they can be used. A large proportion of the cost of PHB, and consequently LCPs, is related to the cost of its chemical synthesis.
PHB production by chemical synthesis requires petrochemical starting materials, whose cost and availability depend on oil. Because of the uncertainty involved in using a process dependent on fossil fuels, making the considerable investment to increase capacity to achieve economies of scale in PHB production is risky without having predetermined markets for the product and therefore is generally not done. Additional cost in the manufacture of PHB by chemical synthesis is due to the hazardous waste stream which is generated. A new, low cost process to manufacture PHB which does not require petrochemical starting materials and does not generate a hazardous waste stream would overcome these problems and greatly reduce the manufacturing costs of LCPs, allowing them to be used more widely. The inventors have been able to develop such a process for the production of PHB using E. coli fermentation.
PHB is produced as a minor metabolite by E. coli, however E. coli is not capable of excreting PHB at levels high enough to be easily detectable or cost-effectively recovered by conventional commercial methods (less than 2 mg of PHB per liter of medium). Therefore, for biological production of PHB by E. coli to be economically feasible, the amount E. coli naturally produces must be dramatically increased.
Typically, increased production of a metabolite is achieved by mutating the microorganisms and selecting improved strains from among them using a screening assay. Naturally occurring (spontaneous) mutations also may be taken advantage of when suitable screening methods are available. Both of these methods require the use of effective screening methods to detect clones having the desired characteristics, a very time-consuming and labor-intensive process both in terms of the development of the screening assays and their execution. In fact, the development and manufacturing costs at this stage can sometimes overreach the potential benefits gained from the improvements in the detected mutant strains.
Another approach to increasing production of a desired metabolite is the manipulation of the particular genes involved in the relevant biological pathway. With the evolution of genetic engineering methods, manipulation and overexpression of selected enzymes have become routine. It is now possible to create a biocatalyst in which all the genes necessary to synthesize a particular product are selected and amplified. However, overexpression of a large group of enzymes adds an extra metabolic burden to the microorganism, sometimes resulting in lower overall production due to poor growth and general metabolism of the cells. In addition, selecting and amplifying each enzyme in the synthesis of a product with multiple steps is costly in both time and labor. Thus, it is important to critically select which genes are to be amplified, how they are overexpressed, and how much they are overexpressed.
PHB is biosynthesized from glucose in E. coli, as shown in FIG. 1. A significant number of steps are required in the metabolic pathway from 3-deoxy-D-arabino-heptulosonate-7-phosphate to chorismic acid. The final and possibly the rate-limiting step in the synthesis, conversion of chorismate to PHB, is catalyzed by chorismate pyruvate lyase (CPL), the expression product of the ubiC gene.
Cloning and characterization of the ubiC gene product and the entire DNA sequence of the ubiC gene has been reported by Siebert et al., Microbiology 140: 897-904 (1994) and by Nichols and Green, Journal of Bacteriology 174 (16): 5309-5316 (1992). Microbial production of PHB using Klebsielia pneumoniae with a plasmid allowing overexpression of CPL also has been reported by Muller et al., Appl. Microbiol. Biotechnol. 43: 985-988 (1995), however, the amount of PHB obtained by this method was only 300 mg/L, insufficient for reasonable commercial production.
In summary, the current methods available to produce PHB either through chemical synthesis, or by biofermentation with K. pneumoniae, suffer from serious disadvantages which make the commercial production of PHB too expensive to allow the general use of LCPs. Therefore, there remains a need for a biofermentation process for the synthesis for 4-hydroxybenzoic acid which can produce large amounts of PHB at a commercially reasonable cost.